Functional Characterization of the Mouse LTC4 Synthase Gene

白三烯(LT)C4合成酶是受体活性半胱氨酰白三烯的母体化合物LTC4生物合成的关键酶。因此，LTC4合成酶可以通过其代谢产物LTD4和LTE4调节这种强有力的脂质介质的生物合成，从而在哮喘和其他炎症性疾病的病理生物学中发挥作用。本项目的目的是确定小鼠LTC4合酶基因的转录调控(Aim 1)；建立LTC4合酶转基因小鼠和LTC4合酶基因干扰小鼠(Aim 2)；并检测这些基因操纵的小鼠对各种炎性侮辱的体内反应(Aim 3)。为了研究小鼠LTC4合成酶基因的转录调控，我们将从先前发现的小鼠LTC4合成酶基因5‘侧翼区的352bp的基因组片段开始。启动子和增强子元件将通过报告结构、突变分析和DNase1超敏试验来确定。反式作用因子将通过凝胶移位和超移位分析来表征。然而，主要的焦点是通过选择性地改变末端生物合成酶LTC4合成酶来评估半胱氨酰白三烯在生理和病理生物学过程中的作用。转基因小鼠将通过原核注射含有完整的人LTC4合成酶基因及其天然启动子的5.5kb基因组片段来产生。用人的特异性寡核苷酸引物对小鼠进行转基因筛查，并通过Southern杂交分析确定它们的基因剂量。基因干扰的小鼠将被构建成一个靶向结构，它包含一个新霉素基因来代替小鼠LTC4合成酶基因的外显子2到外显子4，以及一个胸苷激酶基因在被破坏的LTC4合成酶基因的下游。具有同源重组的双抗性克隆将被用于囊胚注射，以创造嵌合小鼠和随后的基因破坏小鼠。这些基因操作的动物的生长和发育以及它们的BMMC表达LTC4合成酶和产生半胱氨酰白三烯的能力将被检测。对它们的炎症反应的评估将包括对蛋白质敏化和气雾剂攻击的气道高反应性、花生四烯酸引起的炎症、全身过敏反应、旋毛虫感染和非类固醇激素引起的胃溃疡。

Leukotriene (LT)C4 synthase is the pivotal enzyme in the biosynthesis of LTC4, the parent compound of the receptor active cysteinyl leukotrienes. Thus, LTC4 synthase can regulate the biosynthesis of this potent lipid mediator, which contributes to the pathobiology of bronchial asthma and other inflammatory diseases through its metabolites, LTD4 and LTE4. The objectives of this project are to define the transcriptional regulation of the murine LTC4 synthase gene (Aim 1); to generate LTC4 synthase transgenic mice and the LTC4 synthase gene-disrupted mice (Aim 2); and to examine the in vivo responses of these gene manipulated mice to various inflammatory insults (Aim 3). To examine the transcriptional regulation of mouse LTC4 synthase gene, we will start with a previously identified 402-bp genomic fragment of mouse LTC4 synthase gene contining 352-bp of 5' flanking region. Promoter and enhancer elements will be defined by reporter contructs, mutagenic analysis, and by Dnase 1 hypersensitivity assays. Trans-acting factors will be characterized by gel shift and supershift assays. The major focus, however, is to assess the role of the cysteinyl leukotrienes in physiologic and pathobiologic processes by selective alterations of the terminal biosynthetic enzyme, LTC4 synthase. Transgenic mice will be created by pronuclear injection of a 5.5-kb genomic fragment containing the entire human LTC4 synthase gene with its native promoter. Mice will be screened for the transgene by PCR with human specific oligonucleotide primers and their gene dosage determined by Southern blot analysis. The gene-disrupted mice will be created with a targeting construct which contains a neomycin gene in place of exon 2 to exon 4 of mouse LTC4 synthase gene and a thymidine kinase gene downstream of the disrupted LTC4 synthase gene. Double resistence clones with homologous recombination will be used for blastocysts injection to create chimeric mice and subsequently gene-disrupted mice. The growth and development of these gene manipulated animals and the ability of their BMMC to express LTC4 synthase and to generate cysteinyl leukotrienes will be examined. Assessment of their inflammatory responses will include airway hyperreactivity to protein sensitization and aerosol challenge, arachidonic acid induced inflammation, systemic anaphylaxis, T. spiralis infection, and NSAID induced gastric ulcers.